1. Field of the Invention
The field of the invention is improved electrochemical diagnostic reagents useful for instrumented tests for coagulation, immunoassays, and other analytes.
2. Description of the Related Art
There is a wide range of chemical entities (test ligands, test analytes) where rapid identification of the presence and relative levels of the entity are highly important. In medicine, it is often critically important to rapidly identify medical analytes such as hormones, drugs, pathogens, and physiological enzymes. In agricultural areas, it is often important to identify trace levels of contaminants or pathogens, such as harmful bacteria, adulterants, or other undesirable contaminants. In environmental studies, it is often important to rapidly identify trace levels of pollutants. For military applications, identification of trace levels of toxic agents is also important.
As a result of this common need for rapid identification of test ligands, various different rapid detection schemes have been devised. These include general-purpose detection methodologies, such as chromatography and mass spectrometry, and more specialized detection methodologies, such as the various diagnostic chemical methodologies that employ test reagents designed to produce detectable signals upon chemical reaction with the test analyte. The present application is focused on this latter type of rapid chemical test methods.
Although complex automated chemical analyzers exist, using liquid chemical reagents, which can rapidly analyze many different types of test ligand, such devices tend to be expensive, delicate, and often require skilled users. As a result, an alternative approach, using premixed reagents stored in a dry form, and reconstituted by the fluid in the test analyte's sample, has become quite popular in recent years. Such tests are referred to generically as “dry reagent tests”.
There are two basic categories of dry reagent test. Dry reagent tests that produce a detectable change in the electrochemical potential of an electrode are typically referred to as electrochemical dry reagent tests, and dry reagent tests that produce a detectable optical change in the optical characteristics of the reagent (change in color, change in fluorescence, etc.) are typically referred to by the type of optical change used in the assay (e.g. calorimetric tests, fluorescence tests, etc.).
Due to the high demand for simple blood glucose tests for diabetics, electrochemical dry reagent tests have become increasingly popular in recent years. In contrast to optical dry reagent tests, which require both precise optical measuring equipment, and precise ways to translate the optical signal into a final answer, electrochemical tests usually can function with simpler equipment. The need for a precise optical section is eliminated, and the electrochemical signal generated by the reagent can be converted to a final answer using simple and low cost electronic circuits. As a result, electrochemical blood glucose tests have become a multi-billion dollar a year industry. A wide variety of electrochemical methods have been devised, and due to the high economic activity in this space, the technology is now in a well-developed and mature state.
At present, not all analytes can be measured by electrochemical means. This is because in many cases, simple and practical ways to transduce the chemical signal produced by the test reagent-test analyte reaction over to an electrochemical signal capable of being detected at a test reagent electrode has not been identified. As a result, many useful assays, such as immunochemical assays, enzyme substrate assays, and the like must currently be performed using older optical dry reagent technology. Because, in many cases, this technology is not as fully developed as modern dry reagent electrochemical technology, many of these assays are currently being performed using the older, more expensive, and less reliable optical format. Additionally, the lower volume of many of these assays has made it uneconomic to develop improvements, creating many “orphan” tests that have not improved much beyond the original, previous generation, optical technology.
One example of an “orphan” optical dry reagent technology for immunochemical analytes is the Apoenzyme Reactivation Immunoassay (ARIS). The ARIS concept is based upon the formation of a unique type of hybrid molecule. This hybrid molecule consists of an apoenzyme reactivation factor (also called an enzyme “cofactor”, “coenzyme” or “prosthetic group”) that is chemically conjugated to a reagent version of the test ligand (antigen) molecule. This conjugation creates a hybrid molecule containing both an enzyme reactivation factor, and a reagent version of the test ligand (antigen) molecule of interest. The ARIS assay also contains reagent antibodies that bind to this hybrid molecule, and an inactive apoenzyme. In the absence of test analytes, the reagent antibodies bind to the hybrid molecule and prevent the molecule's apoenzyme reactivation factor from reactivating the apoenzyme. In the presence of test analytes, however, the test ligands compete for binding to the reagent antibodies, and displace the hybrid molecules away from the reagent antibodies. The now unbound apoenzyme reactivation factors are now free to reactivate the apoenzyme, which in turn produces a colored reaction product. Although, in some cases, such tests can be observed directly by eye without need of automated instrumentation, direct visual methods have limited accuracy. As a result, optical meters more commonly read such tests. However, as previously discussed, optical metering systems tend to be more complex and more susceptible to inaccuracy, relative to electrochemical metering systems, and thus are less economically attractive. Thus methods to translate optical ARIS immunochemical tests to the more mature electrochemical format are desirable.
A second example of “orphan” dry reagent technology is blood coagulation monitoring assays. Here a variety of dry reagent tests exist, including optical tests, and non-standard electrochemical tests. The later works by principles that are substantially different than the more common enzyme based electrochemical biosensors, and thus are not at the same level of technological maturity as most enzyme based electrochemical biosensors.
At present, all coagulation tests are significantly more expensive than electrochemical blood glucose tests, and all require more complex and sophisticated metering systems. Thus methods to translate blood coagulation tests to the more mature and standard enzyme based electrochemical biosensors are also desirable
Prior art for electrochemically based prothrombin time assays may be found in U.S. Pat. Nos. 6,066,504; 6,060,323; 6,046,051; 6,673,622 by Jina et. al, U.S. Pat. No. 6,352,630 by Frenkel et. al., and U.S. Pat. No. 6,620,310 by O'hara et. al.
Prior art for thrombin substrate based coagulation assays may be found in U.S. Pat. Nos. 5,580,744 and 5,418,141 by Zweig.
Prior art for dry reagent homogeneous apoenzyme reactivation (ARIS) chemistry and immunochemistry can be found in U.S. Pat. Nos. 3,817,837; 4,134,792; 4,213,893; 4,238,565; 4,318,983; 4,495,281 and others.
Prior art for enzyme based electrochemical biosensors for blood glucose can be found a variety of patents, including many assigned to Genetics International, Medisense, E. Heller, & Company, Therasense, Selfcare, Boehringer Mannheim, and others. These include U.S. Pat. Nos. 4,545,382; 4,711,245; 4,758,323; 5,262,035; 5,262,305; 5,264,105; 5,286,362; 5,312,590; 5,320,725; 5,509,410; 5,628,890; 5,682,884; 5,708,247; 5,727,548; 5,820,551; 5,951,836; 6,134,461 and 6,143,164.
Turner, Miller, and Costa, in UK patent application GB 2188728 A, disclose an apoenzyme reactivated electrode system in which antibodies are conjugated to a prosthetic-group-generating enzyme (aminoacylase). Test antigens act as specific binding pairs to cause the conjugated antibodies to bind to an electrode containing an apoenzyme form of an electrically active enzyme GDH. When test antigens are present, the antibody-aminoacylase-enzyme conjugates specifically bind to the test antigens, and carry the prosthetic-group-generating enzyme aminoacylase to the electrode. The antibody coupled aminoacylase enzyme produces a GDH apoenzyme prosthetic group PQQ. These PQQ apoenzyme prosthetic groups, in turn, bind to the electrode-bound GDH apoenzyme, and change the GDH apoenzyme into an electrically active GDH enzyme. This electrically active GDH enzyme in turn produces an electrical signal that is proportional to the amount of the test antigens in the analyte.
Although 728A teaches one specific method of detecting analytes containing antigens (test antigens), 728A fails to teach general methods for detecting enzymatic activity in test samples (i.e. fails to teach how to detect analytes that are enzymatically active). Rather, 728A simply teaches how to detect binding to an antigen. Although 728A's methods include an enzyme (aminoacylase) labeled antibody, 728A is not detecting aminoacylase enzymatic activity in the sample, nor is 728A detecting any other form of sample enzymatic activity. The aminoacylase enzyme is simply used as an antigen (specific binding) detection tool. The aminoacylase enzyme acts by converting a molecule that is not a GDH prosthetic group into a molecule (PQQ) that is a GDH prosthetic group.
In particular, 728A fails to teach how the activity of analyte enzymes that act to cleave polymeric test substrates by hydrolysis, such as proteases, nucleases, and glycosylases, can be directly detected. This is because such analyte enzymes usually do not create apoenzyme prosthetic groups as a reaction product, which is required by 728A's teaching. Since 728A's “specific binding pair” methods only detect antibody binding, (rather than test enzyme activity), 728A's methods will generally be unable to distinguish between situations where the analyte enzyme is present in an inactive or partially active form, and situations where the analyte enzyme is present in an active form.
Since many useful analytical tests, such as blood coagulation, distinguish between active and inactive forms of analyte enzymes, (where the actual molar concentration of the enzymatic protein itself is unchanged), the specific binding pair methods of 728A are unlikely to be effective for this type of application.
Joseph and Madou, in PCT application WO 91/16630 teach another variant of the binding partner method. This method also relies on directly detecting the concentration of the analyte (e.g. number of moles of antigen or protein present in the sample) by specific binding methods, rather than on detecting the enzymatic activity of the analyte enzymes. Thus, just as previously discussed in more detail for Turner et. al., the methods of Joseph will also generally fail to distinguish between analyte enzymes that are present in an inactive form, and analyte enzymes that are present in an active form. Thus the methods of Joseph and Madou also generally fail to perform for enzymatic tests, such as blood coagulation, that must distinguish between active and inactive forms of analyte enzymes.
Thus there remains a need for simple electrochemical methods that can directly detect relative levels of enzymatic activity in a biological sample of interest, as well as detect analyte (test) antigens of interest.